Phosphorylation-dephosphorylation of proteins is one of the most important mechanisms for the regulation of cellular functions. Protein kinase C, a Ca2+/phospholipid-dependent protein kinase, has emerged as a pivotal regulatory element for cell growth, differentiation, gene expression, hormone secretion, cell surface receptor function, and cellular metabloism. This protein kinase can be activated by diacylglycerol, a second messenger generated by signal-induced breakdown of phosphoinositides. In addition, it has been identified as a receptor for tumor-promoting phorbol esters which elicit pleiotropic responses comparable to those by many hormones and growth factors. Three isozymic forms of rat brain protein kinase C have been purified to near homogeneity. Polyclonal and monoclonal antibodies against these enzymes were prepared for the immunochemical characterization. These isozymes are distinguishable by peptide mapping and immunological analysis, indicating that they are products of different genes. The various protein kinase C isozymes were found to be enriched in different regions of rat brain and expressed differentially during brain development. Activation of protein kinase C in cells was studied by using EL4 thymoma cells which can be induced to secret interleukin-2 in response to tumor-promoting phorbol esters. These compounds promote the translocation of protein kinase C from cytosol to the particulate fraction and a rapid degradation of the enzyme. The phorbol ester-induced translocation and degradation of protein kinase C may be early events in the secretion of interleukin-2. In vitro, tryptic degradation of purified protein kinase C generates active forms of the kinase and phorbol ester-binding protein that are active in the absence of Ca2+. The structure-function relationships of protein kinase C were investigated by using monoclonal antibody specific for the phorbol ester-binding domain. This antibody reduces the binding of phorbol ester to the enzyme without inhibiting the kinase activity. Protein kinase C activity was shown to be regulated by autophosphorylation, and the resulting phosphorylated kinase exhibited higher affinity for Ca2+ and phorbol esters.